The goal of this proposal is to understand molecular events regulating the MKK/ERK pathway and mechanisms by which this pathway contributes to the control of proliferation in mammalian cells. During previous funding of this grant, the principal investigator discovered novel regulation and function of MKK/ERK during late S, G2, and mitosis in somatic mammalian cells, distinct from its well-known role in G1/S progression. In the current proposal, it is planned to determine the mechanism of pathway regulation and delineate cellular targets of MKK/ERK, during S/G2IM. The role of MKKJERK during these phases of the cell cycle most likely contributes to maintaining chromosome integrity in mammalian cells, which is known to be destabilized by deregulated Ras and MAPK signaling. Thus, completion of these studies will reveal novel and important insight into mechanisms that induce chromosome instability and promote cancer progression. The specific aims are: (1) Determine the pathway of MKK and ERK activation during mitosis, (2) Define mechanisms for nuclear translocation of MKK during mitosis, (3) Identify cellular targets for the MKK/ERK pathway in S, G2, and M, (4) Examine the regulation and role of ERK activity in microtubule-kinetochore interactions, and (5) Identify kinetochore targets for MKK/ERK signaling. The proposed experiments will utilize biochemical and molecular biological strategies as well as apply new and innovative technologies of proteomics mass spectrometry and optical tweezer microscopy towards these problems in cell regulation. The information to be gained from these studies will provide a unique and unprecedented view of molecular events during mitosis in soluble nuclear and cytosolic compartments about as well as kinetochores, and provide the basis for new insight into how signal transduction processes are integrated with cell cycle regulation.